Monoclonal antibodies to fibroblast growth factor receptor 2

ABSTRACT

The present invention is directed toward a monoclonal antibody to fibroblast growth factor receptor 2, a pharmaceutical composition comprising same, and methods of treatment comprising administering such a pharmaceutical composition to a patient.

CROSS-REFERENCES TO RELATED APPLICATIONS

This applications claims the benefit under 35 U.S.C. §119(e) of U.S.patent application Ser. No. 61/112,686 filed Nov. 7, 2008 and U.S.patent application Ser. No. 61/164,870 filed Mar. 30, 2009, which areherewith incorporated in their entirety for all purposes.

STATEMENT OF GOVERNMENT INTEREST

The invention described in this application was made in part with fundsprovided by Grant 5R44 CA101283-03 from the National Institutes ofHealth. The US Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to the combination of monoclonalantibody (mAb) and recombinant DNA technologies for developing novelbiologics, and more particularly, for example, to the production ofmonoclonal antibodies that bind to and neutralize Fibroblast GrowthFactor Receptor 2.

BACKGROUND OF THE INVENTION

There are 22 known members of the Fibroblast Growth Factor (FGF) family,ranging in size from 17 to 34 kDa and sharing an internal core region ofsimilarity, which can be grouped into 7 subfamilies based on theirsimilarity in activity and sequence (Ornitz et al., Genome Biol.2:3005.1, 2001). For example, the FGF1 subgroup consists of theprototypical FGFs, FGF1 (acidic FGF) and FGF2 (basic FGF); the FGF4subgroup consists of FGF4, FGF5 and FGF6; and the FGF7 subfamilyconsists of FGF3, FGF7, FGF10 and FGF22 (Zhang et al., J. Biol. Chem.281:15694, 2006).

One form of FGF2 is an 18 kDa non-glycosylated polypeptide consisting of146 amino acids derived from a 155 aa precursor (Ornitz et al., GenomeBiol. 2:3005.1, 2001; Okada-Ban et al., Int. J. Biochem. Cell. Biol.32:263, 2000). An exemplary sequence for a human 146 amino acid FGF2 isprovided in SEQ ID NO:4 of US20020115603. Unlike most other FGFs, FGF2does not encode a signal sequence for secretion, but the 18 kDa form canbe secreted by an unconventional energy-dependent pathway independent ofthe ER-Golgi complex. The other FGF1 subfamily member, FGF1 itself, hassize and structure similar to FGF2 and also lacks a signal sequence butcan be secreted. Another FGF of interest here is FGF7, also calledkeratinocyte growth factor (KGF), which is produced by cells ofmesenchymal origin and stimulates epithelial cell proliferation (Finchet al., Adv. Cancer Res. 91:69, 2004; Finch et al., J. Natl. CancerInst. 98:812, 2006). KGF is expressed in a number of organs includinglung, prostate, mammary, digestive tract and skin and is implicated inorgan development and repair of cutaneous wounds (Cho et al., Am. J.Pathol. 170:1964, 2007).

The FGF family members bind to only four known tyrosine kinasereceptors, Fibroblast Growth Factor Receptors 1-4 (FGFR1-4) and theirisoforms, with the various FGFs binding the different FGFRs to varyingextents (Zhang et al., J. Biol. Chem. 281:15694, 2006). A proteinsequence of human FGFR2 is provided in, e.g., GenBank Locus AF487553.Each FGFR consists of an extracellular domain (ECD) comprising threeimmunoglobulin (Ig)-like domains (D1, D2 and D3), a single transmembranehelix, and an intracellular catalytic kinase domain (Mohammadi et al.,Cytokine Growth Factor Revs, 16:107, 2005) as illustrated in FIG. 1.There is a contiguous stretch of acidic amino acids in the linkerbetween D1 and D2 called the “acid box” (AB). The region containing D1and AB is believed to be involved in autoinhibition of the receptor,which is relieved by binding to ligand. The FGFRs are characterized bymultiple alternative splicing of their mRNAs, leading to a variety ofisoforms (Ornitz et al., J. Biol. Chem. 271:15292, 1996; see alsoSwiss-Prot P21802 and isoforms P21802-1 to -20 for sequences of FGFR2and its isoforms). Notably, there are forms containing all three Igdomains (α isoform) or only the two Ig domains D2 and D3 domains withoutD1 (β isoform). Of particular importance in FGFR1-FGFR3, while all formscontain the first half of D3 denoted IIIa, two alternative exons can beutilized for the second half of D3, leading to IIIb and IIIc forms. ForFGFR2, these are respectively denoted FGFR2IIIb and FGFR2IIIc (or justFGFR2b and FGFR2c); the corresponding beta forms are denotedFGFR2(beta)IIIb and FGFR2(beta)IIIc. The FGFR2IIIb form of FGFR2 (alsodenoted K-sam-II) is a high affinity receptor for both FGF1 and KGFwhereas FGFR2IIIc (also denoted K-sam-I) binds both FGF1 and FGF2 wellbut does not bind KGF (Miki et al., Proc. Natl. Acad. Sci. USA 89:246,1992). Indeed, FGFR2IIIb is the only receptor for KGF (Ornitz et al.,1996, op. cit.) and is therefore also designated KGFR.

The FGFRs and their isoforms are differentially expressed in varioustissues. Notably, FGFR2IIIb (and the IIIb forms of FGFR1 and FGFR3) areexpressed in epithelial tissues, while FGFRIIIc is expressed inmesenchymal tissues (Duan et al., J. Biol. Chem. 267:16076, 1992; Ornitzet al., 1996, op. cit.). Certain of the FGF ligands of these receptorshave an opposite pattern of expression. Thus, FGF3 subfamily membersincluding FGF7 (KGF) bind only to FGFRIIIb (Zhang et al., op. cit.) andare expressed in mesenchymal tissues so may be paracrine effectors ofepithelial cells (Ornitz et al., 1996, op. cit.). In contrast, the FGF4subfamily members FGF4-6 bind to FGFR2IIIc and are expressed in bothepithelial and mesenchymal lineages so may have either autocrine orparacrine functions. Because of the expression patterns of the isoformsof FGFR2 and their ligands, FGFR2 plays a role in epithelial-mesynchymalinteractions (Finch et al., Dev. Dyn. 203:223, 1995), so it is notsurprising that knock-out of FGFR2IIIb in mice leads to severe embryonicdefects and lethality (De Moerlooze et al., Development 127:483, 2000).

In addition to binding FGFR1-4 with high affinity, the FGFs bind toheparin sulfate proteoglycans (HSPG) with lower affinity. In fact,binding of FGF to heparin/heparin sulfate (HS) on the cell surface isrequired for signalling through the FGFRs. The interaction of FGF,especially FGF2, with FGFR and heparin has been extensively studied byX-ray crystallography and mutational analysis, and it is now believedthat heparin/HS participates in the formation of a symmetric 2:2FGF-FGFR dimer (Mohammadi et al., 2005), leading to receptor activation,autophophorylation and signal transduction.

The FGFs mediate a variety of responses in various cell types includingproliferation, migration and differentiation, especially duringembryonic development (Ornitz et al., op. cit.), and in the adult areinvolved in tissue homeostasis and repair. For example, FGF2 stimulatesproliferation of (i.e., is mitogenic for) certain cells includingfibroblasts and endothelial cells and is an anti-apoptotic survivalfactor for certain cells such as neural cells (Okada-Ban, op. cit.).FGF2 also stimulates differentiation (morphogenesis) and migration(motility) of endothelial cells (Dow et al., Urology 55:800, 2000).Several FGFs, especially FGF1 and FGF2, are potent angiogenic factors(Presta et al., Cytokine and Growth Factor Rev. 16:159, 2005).

The importance of the FGF system in development has been highlighted bythe discovery of numerous mutations in FGFR1-3 associated with humancongenital skeletal disorders including the craniosynostosis syndromes(premature fusion of the cranial sutures) (Wilkie et al., CytokineGrowth Factor Revs 16:187, 2005). These genetic diseases are usuallydominant because the associated mutations lead to gain-of-function,often by facilitating receptor dimerization. Notably, the severecraniosynostosis disorder Apert syndrome (AS) is associated with one oftwo mutations (Ser-252→Trp or Pro-253→Arg) in the conserved D2-D3 linkerregion of FGFR2 that increase ligand binding affinity (Ibrahimi et al.,Proc. Natl. Acad. Sci USA 98:7182, 2001).

FGF2 and other FGFs have been reported to play a role in cancer, both bystimulating angiogenesis and tumor cells directly (Grose et al.,Cytokine Growth Factor Revs. 16:179, 2005; Presta et al., op cit.).FGFR2IIIb RNA is expressed in many types of tumors (Finch et al., J.Natl, Cancer Inst. 98:812, 2006), often as a consequence of itsexpression in the corresponding normal tissues (Orr-Urtreger et al.,Dev. Biol. 158:475, 1993). KGF (FGF7) and KGFR (FGFR2IIIb) areoverexpressed in many pancreatic cancers (Ishiwata et al., Am. J.Pathol. 153: 213, 1998), and their coexpression correlates with poorprognosis (Cho et al., Am. J. Pathol. 170:1964, 2007). Somatic mutationsof the FGFR2 gene were found in 12% of a large panel of endometrial(uterine) carcinomas, and in several tested cases were required fortumor cell survival (Dutt et al., Proc. Natl. Acad. Sci. USA 105:8713,2008). In two tumors the FGFR2 mutation was found to be the same S252Wsubstitution associated with Apert syndrome. Amplification andoverexpression of FGFR2 is strongly associated with theundifferentiated, diffuse type of gastric cancer, which has aparticularly poor prognosis, and inhibition of the FGFR2 activity bysmall molecule compounds potently inhibited proliferation of such cancercells (Kunii et al., Cancer Res. 68:2340, 2008; Nakamura et al.,Gastroenterol. 131:1530, 2006). FGFR2IIIb RNA was expressed in 16/20epithelial ovarian cancers (EOGs) but not in normal ovarian surfaceepithelium (Steele et al., Oncogene 20:5878, 2001); and the FGFR2IIIbligands FGF1, FGF7 and FGF10 induced proliferation, motility andprotection form cell death in EOC cell lines (Steele et al., GrowthFactors 24:45, 2006), suggesting that FGFR2IIIb may contribute to themalignant phenotype in ovarian cancer.

Only a limited number of monoclonal antibodies to FGFR2 have beenreported. Fortin et al. (J. Neurosci. 25:7470, 2005) reported a blockingantibody to FGFR2, and Wei et al. (Hybridoma 25: 115, 2006) developedtwo mAbs specific for the IIIb form of FGFR2 (i.e, KGFR) that inhibitedKGF-induced cell proliferation. Yayon et al. (W02007/144893, 2006)disclosed an inhibitory mAb that binds both FGFR2 and FGFR3. R&D Systemshas marketed since 2005 an anti-FGFR2 mAb that neutralizes activity intheir assay, with preference for the IIIb form. However, there have beenno reports of anti-tumor activity of antibodies against FGFR2 in vivo.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a monoclonal antibody (mAb) tohuman fibroblast growth factor receptor 2 (FGFR2) that inhibits growthof a human tumor xenograft in a mouse. The mAb may inhibit at least one,and preferably several or all biological activities of the receptor,including binding to the receptor by FGF2. The mAb can bind to either orboth of the FGFR2IIIb and FGFRIIIc forms of the receptor, e.g., toFGFR2IIIb but not to FGFR2IIIc Preferably, the mAb of the invention isgenetically engineered, for example, chimeric, humanized or human.Exemplary antibodies are GAL-FR21, GAL-FR22, and GAL-FR23 and theirchimeric and humanized forms, and mAbs which have the same epitope orcompete for binding with one of these mAbs. In another embodiment, apharmaceutical composition comprising a genetically engineeredanti-FGFR2 antibody, e.g., chimeric or humanized GAL-FR21, GAL-FR22, andGAL-FR23, is provided. In a third embodiment, the pharmaceuticalcomposition is administered to a patient to treat cancer or otherdisease, for example gastric cancer.

Exemplified humanized antibodies comprise a humanized light chaincomprising CDRs from the sequence in FIG. 13A (GAL-FR21) and a humanizedheavy chain comprising CDRs from the sequence of FIG. 13B (GAL-FR21), orcomprise a humanized light chain comprising CDRs from the sequence inFIG. 16A (GAL-FR22) and a humanized heavy chain comprising CDRs from thesequence of FIG. 16B (GAL-FR22). Some humanized antibodies comprise thethree light chain CDRs shown in FIG. 13A (GAL-FR21) and the three heavychain CDRs shown in FIG. 13B (GAL-FR21), or comprise the three lightchain CDRs shown in FIG. 16A (GAL-FR22) and the three heavy chain CDRsshown in FIG. 16B (GAL-FR22). Optionally, the light chain variableregion has at least 95% sequence identity to the sequence shown in FIG.13A (HuGAL-FR21) and the heavy chain variable region has at least 95%sequence identity to the sequence shown in FIG. 13B (HuGAL-FR21). Insome such antibodies, residues H27, H28, H30, H48, and H67 by Kabatnumbering are occupied by the residue occupying the correspondingposition of the heavy chain shown in FIG. 13B (GAL-FR21). A preferredhumanized antibody comprises a light chain variable region having thesequence shown in FIG. 13A (HuGAL-FR21) and a heavy chain variableregion having the sequence shown in FIG. 13B (HuGAL-FR21).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of the structure of FGFR2, showing the threeIg-like domains (D1, D2 and D3), transmembrane domain (black box), andintracellular kinase domain. The heparin binding site (HBS), acid box(AB) and alternative IIIb/IIIc partial domains are indicated. N=aminoterminus, C=carboxy terminus.

FIG. 2. Summary of properties of the anti-FGFR2 mAbs GAL-FR21, GAL-FG22,GAL-FR23 as described under Examples.

FIG. 3. Binding ELISA of mAbs GAL-FR21, GAL-FR22 and GAL-FR23 andnegative control mIgG to FGFR2IIIb.

FIG. 4. Binding ELISA of mAbs GAL-FR21, GAL-FR22 and GAL-FR23 andnegative control mouse mAb 5G8 to each of the four forms ofFGFR2-FGFR2IIIb, FGFR2(beta)IIIb, FGFR2(IIIc) and FGFR2(beta)IIIc—asfusion proteins with Fc. A fixed concentration of each mAb was used inthe assay.

FIG. 5. Competitive binding ELISA to FGFR2IIIb of each of the mAbsGAL-FR21, GAL-FR22 and GAL-FR23 and negative control mouse mAb 5G8against the mAbs in biotinylated form. A 100:1 ratio of unlabeled tobiotinylated mAb was used.

FIG. 6. Flow cytometry of binding of the mAbs GAL-FR21, GAL-FR22 andGAL-FR23 and negative control mAb to FGFR2IIIb on SNU-16 and KATO IIIcells.

FIG. 7. Flow cytometry of the mAbs GAL-FR21, GAL-FR22 and GAL-FR23 andnegative control mAb binding to 293F cells transfected with FGFR2IIIc orFGFR2IIIb(S252W).

FIGS. 8A and B. (A) ELISA assay measuring inhibition of binding of FGF1(upper panel) and FGF2 (lower panel) to FGFR2IIIb by mAbs GAL-FR21,GAL-FR22 and GAL-FR23. (B) ELISA assay measuring inhibition of bindingof FGF7 (upper panel) and FGF10 (lower panel) to FGFR2IIIb by mAbsGAL-FR21 and GAL-FR22. For (A) and (B), mIgG is negative control mousemAb.

FIG. 9. Growth of SNU-16 human gastric tumor xenografts in mice treatedwith PBS alone, GAL-FR21, GAL-FR23 or FR2bC 54.8.11 (upper panel) orwith PBS, GAL-FR22 or FR2bC 54.8.11 (lower panel). The mAbs wereadministered at 20 μg twice per week, about 5 mice per group.

FIG. 10. Growth of SNU-16 (A, upper panel) or OCUM-2M (B, lower panel)human gastric tumor xenografts in mice treated with PBS alone, GAL-FR21or GAL-FR22.

FIG. 11. Binding ELISA of GAL-FR21 (upper panel) or GAL-FR22 (lowerpanel) to human and mouse FGFR2IIIb.

FIG. 12. Binding ELISA of GAL-FR21 (upper panel) or GAL-FR22 (lowerpanel) to human and cynomolgus monkey FGFR2IIIb.

FIG. 13. Amino acid sequences of the HuGAL-FR21 light chain (A) andheavy chain (B) mature variable regions are shown aligned with mouseGAL-FR21 and human acceptor V regions. The CDRs are underlined in theGAL-FR21 sequences, and the amino acids substituted with mouse aminoacids are double underlined in the HuGAL-FR21 sequences. The 1-letteramino acid code and Kabat numbering system are used for both the lightand heavy chain.

FIG. 14. Amino acid sequences of the entire mature HuGAL-FR21 antibodylight chain (A) and heavy chain (B). The first amino acid on each lineis numbered; the numbering is sequential. In the light chain, the firstamino acid of the OK region is underlined, and in the heavy chain, thefirst amino acids of the CH1, hinge, CH2 and CH3 regions are underlined.

FIG. 15. Competitive binding of humanized HuGAL-FR21 and mouse GAL-FR21mAbs and control human antibody hIgG, conducted as described in thespecification.

FIG. 16. Amino acid sequences of the HuGAL-FR22 light chain (A) andheavy chain (B) mature variable regions, with the CDRs underlined. The1-letter amino acid code and Kabat numbering system are used for boththe light and heavy chain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides anti-FGFR2 monoclonal antibodies (mAbs) thatinhibit biological activities of FGFR2 and/or inhibit growth of anFGFR2-expressing tumor xenograft in a mouse, pharmaceutical compositionscomprising the mAbs, and methods of using them for the treatment ofdisease.

1. Antibodies

Antibodies are very large, complex molecules (molecular weight of˜150,000 or about 1320 amino acids) with intricate internal structure. Anatural antibody molecule contains two identical pairs of polypeptidechains, each pair having one light chain and one heavy chain. Each lightchain and heavy chain in turn consists of two regions: a variable (“V”)region involved in binding the target antigen, and a constant (“C”)region that interacts with other components of the immune system. Thelight and heavy chain variable regions come together in 3-dimensionalspace to form a variable region that binds the antigen (for example, areceptor on the surface of a cell). Within each light or heavy chainvariable region, there are three short segments (averaging 10 aminoacids in length) called the complementarity determining regions(“CDRs”). The six CDRs in an antibody variable domain (three from thelight chain and three from the heavy chain) fold up together in 3-Dspace to form the actual antibody binding site which locks onto thetarget antigen. The position and length of the CDRs have been preciselydefined by Kabat, E. et al., Sequences of Proteins of ImmunologicalInterest, U.S. Department of Health and Human Services, 1983, 1987. Thepart of a variable region not contained in the CDRs is called theframework, which forms the environment for the CDRs.

A humanized antibody is a genetically engineered antibody in which theCDRs from a mouse antibody (“donor antibody”, which can also be rat,hamster or other non-human species) are grafted onto a human antibody(“acceptor antibody”). The sequence of the acceptor antibody can be, forexample, a mature human antibody sequence, a consensus sequence of humanantibody sequences, or a germline region sequence. Thus, a humanizedantibody is an antibody having CDRs from a donor antibody and variableregion framework and constant regions from a human antibody. Inaddition, in order to retain high binding affinity, at least one of twoadditional structural elements can be employed. See, U.S. Pat. Nos.5,530,101 and 5,585,089, incorporated herein by reference, which providedetailed instructions for construction of humanized antibodies. Althoughhumanized antibodies often incorporate all six CDRs (preferably asdefined by Kabat, but often also including hypervariable loop H1 asdefined by Chothia) from a mouse antibody, they can also be made withless than the complete CDRs from a mouse antibody (e.g., Pascalis etal., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of MolecularBiology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol.36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164:1432-1441,2000).

Similarly, it may be necessary to incorporate only part of the CDRs,namely the subset of CDR residues required for binding, termed the SDRs,into the humanized antibody. CDR residues not contacting antigen and notin the SDRs can be identified based on previous studies (for exampleresidues H60-H65 in CDR H2 are often not required), from regions ofKabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol.Biol. 196:901, 1987), by molecular modeling and/or empirically, or asdescribed in Gonzales et al., Mol. Immunol. 41: 863, 2004. In suchhumanized antibodies at positions in which one or more donor CDRresidues is absent, the amino acid occupying the position can be anamino acid occupying the corresponding position (by Kabat numbering) inthe acceptor antibody sequence. The number of such substitutions ofacceptor for donor amino acids in the CDRs to include reflects a balanceof competing considerations. Such substitutions are potentiallyadvantageous in decreasing the number of mouse amino acids in ahumanized antibody and consequently decreasing potential immunogenicity.However, substitutions can also cause changes of affinity, andsignificant reductions in affinity are preferably avoided. Positions forsubstitution within CDRs and amino acids to substitute can also beselected empirically.

Thus, typically a humanized antibody comprises (i) a light chaincomprising CDRs (often three CDRs) from a mouse antibody, e.g.,GAL-FR21, a human variable region framework, and a human constantregion; and (ii) a heavy chain comprising CDRs (often three CDRs) fromthe mouse antibody, e.g., GAL-FR21, a human variable region framework,and a human constant region. The light and heavy chain variable regionframeworks may each be a mature human antibody variable region frameworksequence, a germline variable region framework sequence (combined with aJ region sequence), or a consensus sequence of human antibody variableregion framework sequences.

In the first structural element mentioned above, the framework of theheavy chain variable region of the humanized antibody is chosen to havemaximal sequence identity (between 65% and 95%) with the framework ofthe heavy chain variable region of the donor antibody, by suitablyselecting the acceptor antibody from among the many known humanantibodies. In the second structural element, in constructing thehumanized antibody, selected amino acids in the framework of the humanacceptor antibody (outside the CDRs) are replaced with correspondingamino acids from the donor antibody, in accordance with specified rules.Specifically, the amino acids to be replaced in the framework are chosenon the basis of their ability to interact with the CDRs. For example,the replaced amino acids can be adjacent to a CDR in the donor antibodysequence or within 4-6 angstroms of a CDR in the humanized antibody asmeasured in 3-dimensional space.

A chimeric antibody is an antibody in which the variable region of amouse (or other rodent) antibody is combined with the constant region ofa human antibody; their construction by means of genetic engineering iswell-known. Such antibodies retain the binding specificity of the mouseantibody, while being about two-thirds human. The proportion of nonhumansequence present in mouse, chimeric and humanized antibodies suggeststhat the immunogenicity of chimeric antibodies is intermediate betweenmouse and humanized antibodies. Other types of genetically engineeredantibodies that may have reduced immunogenicity relative to mouseantibodies include human antibodies made using phage display methods(Dower et al., WO91/17271; McCafferty et al., WO92/001047; Winter,WO92/20791; and Winter, FEBS Lett. 23:92, 1998, each of which isincorporated herein by reference) or using transgenic animals (Lonberget al., WO93/12227; Kucherlapati WO91/10741, each of which isincorporated herein by reference).

As used herein, the term “human-like” antibody refers to a mAb in whicha substantial portion of the amino acid sequence of one or both chains(e.g., about 50% or more) originates from human immunoglobulin genes.Hence, human-like antibodies encompass but are not limited to chimeric,humanized and human antibodies. As used herein, a mAb with“reduced-immunogenicity” is one expected to have significantly lessimmunogenicity than a mouse antibody when administered to humanpatients. Such antibodies encompass chimeric, humanized and human mAbsas well as mAbs made by replacing specific amino acids in mouseantibodies that may contribute to B- or T-cell epitopes, for exampleexposed residues (Padlan, Mol. Immunol. 28:489, 1991). As used herein, a“genetically engineered” mAb is one for which the genes have beenconstructed or put in an unnatural environment (e.g., human genes in amouse or on a bacteriophage) with the help of recombinant DNAtechniques, and would therefore, e.g., not encompass a mouse mAb madewith conventional hybridoma technology.

Other approaches to design humanized antibodies may also be used toachieve the same result as the methods in U.S. Pat. Nos. 5,530,101 and5,585,089 described above, for example, “superhumanization” (see Tan etal. J. Immunol. 169: 1119, 2002, and U.S. Pat. No. 6,881,557) or themethod of Studnicak et al., Protein Eng. 7:805, 1994. Moreover, otherapproaches to produce genetically engineered, reduced-immunogenicitymAbs include “reshaping”, “hyperchimerization” andveneering/resurfacing, as described, e.g., in Vaswami et al., Annals ofAllergy, Asthma and Immunology 81:105, 1998; Roguska et al. Protein Eng.9:895, 1996; and U.S. Pat. Nos. 6,072,035 and 5,639,641.

The epitope of a mAb is the region of its antigen to which the mAbbinds. Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1x, 5x, 10x, 20x or 100x excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res. 50:1495, 1990). Alternatively, two antibodies have the sameepitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other. Two antibodies have overlapping epitopes if some aminoacid mutations that reduce or eliminate binding of one antibody reduceor eliminate binding of the other.

2. Anti-FGFR2 Antibodies

A monoclonal antibody (mAb) that binds FGFR2 (i.e., an anti-FGFR2 mAb)is said to neutralize FGFR2, or to be neutralizing (or inhibitory orantagonist), if the binding partially or completely inhibits one or morebiological activities of FGFR2. Among the biological activities of FGFR2that a neutralizing antibody may inhibit or block is the ability ofFGFR2 to bind to one or more or all of its FGF ligands, e.g. FGF1 and/orFGF2. For FGFRIIIb these ligands encompass FGF1, FGF7 (KGF) and theother members of the FGF7 subfamily FGF3, FGF10 and FGF22. For FGFRIIIcthese ligands encompass FGF1 and FGF2; FGF4 and the other members of theFGF4 subfamily FGF5 and FGF6; FGF8 and the other members of the FGF8subfamily FGF17 and FGF18; and FGF9 and the other members of the FGF9subfamily FGF16 and FGF20. Another important activity of FGFR2 that maybe inhibited by a neutralizing anti-FGFR2 mAb is stimulation ofproliferation of cells, e.g., epithelial or endothelial cells,fibroblasts, cells such as Ba/F3 cells into which FGFR2 has beentransfected, and various human tumor cells. Other activities inhibitableby a neutralizing anti-FGFR2 mAb are stimulation of differentiation andmigration of cells such as endothelial cells, and induction ofangiogenesis, for example as measured by stimulation of human vascularendothelial cell (HUVEC) proliferation or tube formation or by inductionof blood vessels when applied to the chick embryo chorioallantoicmembrane (CAM). Usually, the neutralizing mAb inhibits these activitieswhen induced by one or more of the FGFs listed above. Similarly, the mAbpreferably inhibits all or part of the signal transduction pathwaystimulated by binding of an FGF ligand to FGFR2 (Dailey et al., CytokineGrowth Factor Revs 16:233, 2005), e.g., phosphorylation of FGFR2 anddownstream MAP kinases.

A neutralizing mAb of the invention at a concentration of, e.g., 0.01,0.1, 0.5, 1, 2, 5, 10, 20 or 50 μg/ml inhibits a biological function ofFGFR2 by about at least 50% but preferably 75%, more preferably by 90%or 95% or even 99%, and most preferably approximately 100% (essentiallycompletely or indistinguishably from a negative control lacking FGFR2)as assayed by methods described under Examples or known in the art.Typically, the extent of inhibition is measured when the amount of FGFligand used is just sufficient to fully stimulate the biologicalactivity, or is 1, 2, or 5 ng/ml or 0.01, 0.02, 0.05, 0.1, 0.5, 1, 3 or10 μg/ml. Preferably, the mAb is neutralizing, i.e., inhibits thebiological activity, when used as a single agent, but optionally 2 mAbscan be used together to give inhibition. Most preferably, the mAbneutralizes not just one but two, three or several of the biologicalactivities listed above; for purposes herein, an anti-FGFR2 mAb thatused as a single agent neutralizes all the biological activities ofFGFR2 is called “fully neutralizing”, and such mAbs are most preferable.

The instant invention provides neutralizing mAbs that bind FGFR2IIIb butbind less well or not detectably to FGFRIIIc, or alternatively bind toFGFR2IIIc but less well or not detectably to FGFRIIIb, or in a thirdalternative bind to both FGFR2IIIb and FGFR2IIIc, and the use of any ofthese types of antibodies in a pharmaceutical composition, especiallyfor the treatment of cancer or other diseases. The invention alsoprovides mAbs, either neutralizing or non-neutralizing, that bind FGFR2in one or more of its forms and inhibit, preferably completely, growthof a tumor xenograft that expresses FGFR2, e.g., a SNU-16 or OCUM-2Mxenograft. Such a mAb may inhibit tumor growth by, e.g., transmitting anegative growth signal or a pro-apoptotic signal through FGFR2. MAbs ofthe invention are preferably specific for FGFR2 or bind itpreferentially, that is they do not bind, or only bind to a much lesserextent (e.g., at least 10-fold less), proteins that are related to FGFR2such as the other FGF receptors FGFR1, FGFR3 and FGFR4 as well as othermembrane receptor tyrosine kinases. On the other hand, in someinstances, mAbs that bind one or more of the other FGF receptors inaddition to FGFR2 are preferred. MAbs of the invention typically have abinding affinity (association constant K_(a)) for FGFR2 of at least 10⁷M⁻¹ but preferably 10⁸ M⁻¹ or higher, and most preferably 10⁹ M⁻¹ orhigher or even 10¹⁰ M⁻¹ or higher. MAbs showing differential orpreferential binding to one form of FGFR or FGFR2 over another,preferably show a preference of at least five, ten or hundred foldbetween the forms, e.g., as measured by K_(a). Lack of binding betweenan antibody and antigen (i.e., the antibody does not bind the antigen)means any signal from an attempted binding reaction between the two isindistinguishable from a negative control, e.g., in which antibody orantigen is absent or replaced by an inactive agent.

Some mAbs of the invention bind both human FGFR2 and mouse FGFR2, orbind human FGFR2 and one, two or more or all of mouse, rat, rabbit,chicken, dog and/or monkey (e.g., cynomolgus monkey) FGFR2. In someinstances, the mAb binds mouse FGFR2 (e.g., mouse FGFR2IIIb) with anaffinity (i.e., K_(a)) within 2, 10 or 100-fold of that of the affinityfor human FGFR2; similarly the mAb may bind cynomolgus monkey and/orchimpanzee FGFR2 (e.g., FGFR2IIIb) with an affinity within 2 or 10-foldof that of the affinity for human FGFR2 or even substantially the sameas or indistinguishably from binding to human FGFR2 (i.e., withinexperimental error). Other mAbs are specific for only human FGFR2.

MAbs of the invention include anti-FGFR2 antibodies in their naturaltetrameric form (2 light chains and 2 heavy chains) and may be of any ofthe known isotypes IgG, IgA, IgM, IgD and IgE and their subtypes, i.e.,human IgG1, IgG2, IgG3, IgG4 and mouse IgG1, IgG2a, IgG2b, and IgG3. ThemAbs of the invention also include fragments of antibodies such as Fv,Fab and F(ab′)₂; bifunctional hybrid antibodies (e.g., Lanzavecchia etal., Eur. J. Immunol. 17:105, 1987), single-chain antibodies (Huston etal., Proc. Natl. Acad. Sci. USA 85:5879, 1988; Bird et al., Science242:423, 1988); single-arm antibodies (Nguyen et al., Cancer Gene Ther.10:840, 2003); and antibodies with altered constant regions (e.g., U.S.Pat. No. 5,624,821). The mAbs may be of animal (e.g., mouse, rat,hamster or chicken) origin, or they may be genetically engineered.Rodent mAbs are made by standard methods, comprising multipleimmunization with FGFR2 in appropriate adjuvant i.p., i.v., or into thefootpad, followed by extraction of spleen or lymph node cells and fusionwith a suitable immortalized cell line, and then selection forhybridomas that produce antibody binding to FGFR2, e.g., see underExamples. Chimeric and humanized mAbs, made by art-known methodsmentioned supra, are preferred embodiments of the invention. Humanantibodies made, e.g., by phage display or transgenic mice methods arealso preferred (see e.g., Dower et al., McCafferty et al., Winter,Lonberg et al., Kucherlapati, supra).

The anti-FGFR2 mAbs GAL-FR21, GAL-FR22 and GAL-FR23 described below areexamples of the invention. Once a single, archtypal anti-FGFR2 mAb, forexample GAL-FR21, has been isolated that has the desired propertiesdescribed herein of neutralizing FGFR2, it is straightforward togenerate other mAbs with similar properties, e.g., having the sameepitope, by using art-known methods. For example, mice may be immunizedwith FGFR2 as described above, hybridomas produced, and the resultingmAbs screened for the ability to compete with the archtypal mAb forbinding to FGFR2. Mice can also be immunized with a smaller fragment ofFGFR2 containing the epitope to which the archtypal mAb binds. Theepitope can be localized by, e.g., screening for binding to a series ofoverlapping peptides spanning FGFR2. Alternatively, the method ofJespers et al., Biotechnology 12:899, 1994 may be used to guide theselection of mAbs having the same epitope and therefore similarproperties to the archtypal mAb, e.g., GAL-FR21. Using phage display,first the heavy chain of the archtypal antibody is paired with arepertoire of (preferably human) light chains to select an FGFR2-bindingmAb, and then the new light chain is paired with a repertoire of(preferably human) heavy chains to select a (preferably human)FGFR2-binding mAb having the same epitope as the archtypal mAb.Alternatively variants of, e.g., GAL-FR21 can be obtained by mutagenesisof cDNA encoding the heavy and light chains of GAL-FR21.

MAbs with the same or overlapping epitope as GAL-FR21, GAL-FG22 orGAL-FR23, e.g., that compete for binding to FGFR2 with the respectivemAb, provide other examples of the invention. A chimeric or humanizedform of GAL-FR21, GAL-FG22 or GAL-FR23 is an especially preferredembodiment. MAbs that are 90%, 95% or 99% identical to GAL-FR21,GAL-FG22 or GAL-FR23 in amino acid sequence of the heavy and/or lightchain variable regions (not including the signal sequence) and maintainits functional properties, and/or which differ from the respective mAbby a small number of functionally inconsequential amino acidsubstitutions (e.g., conservative substitutions), deletions, orinsertions are also included in the invention. MAbs having at least oneand preferably all six CDR(s) that are 90%, 95% or 99% or 100% identicalto corresponding CDRs of GAL-FR21, GAL-FG22 or GAL-FR23 are alsoincluded. Here, as elsewhere in this application, percentage sequenceidentities are determined with antibody sequences maximally aligned bythe Kabat numbering convention. After alignment, if a subject antibodyregion (e.g., the entire mature variable region of a heavy or lightchain) is being compared with the same region of a reference antibody,the percentage sequence identity between the subject and referenceantibody regions is the number of positions occupied by the same aminoacid in both the subject and reference antibody region divided by thetotal number of aligned positions of the two regions, with gaps notcounted, multiplied by 100 to convert to percentage.

For purposes of classifying amino acid substitutions as conservative ornonconservative, amino acids may be grouped as follows: Group I(hydrophobic side chains); met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acid side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain conformation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same group.Non-conservative substitutions constitute exchanging a member of one ofthese groups for a member of another.

Native mAbs of the invention may be produced from their hybridomas.Genetically engineered mAbs, e.g., chimeric or humanized mAbs, may beexpressed by a variety of art-known methods. For example, genes encodingtheir light and heavy chain V regions may be synthesized fromoverlapping oligonucleotides and inserted together with available Cregions into expression vectors (e.g., commercially available fromInvitrogen) that provide the necessary regulatory regions, e.g.,promoters, enhancers, poly A sites, etc. Use of the CMVpromoter-enhancer is preferred. The expression vectors may then betransfected using various well-known methods such as lipofection orelectroporation into a variety of mammalian cell lines such as CHO ornon-producing myelomas including Sp2/0 and NS0, and cells expressing theantibodies selected by appropriate antibiotic selection. See, e.g., U.S.Pat. No. 5,530,101. Larger amounts of antibody may be produced bygrowing the cells in commercially available bioreactors.

Once expressed, the mAbs or other antibodies of the invention may bepurified according to standard procedures of the art such asmicrofiltration, ultrafiltration, protein A or G affinitychromatography, size exclusion chromatography, anion exchangechromatography, cation exchange chromatography and/or other forms ofaffinity chromatography based on organic dyes or the like. Substantiallypure antibodies of at least about 90 or 95% w/w homogeneity arepreferred, and 98% or 99% w/w or more homogeneity most preferred, forpharmaceutical uses.

3. Treatment Methods

The invention provides methods of treatment in which the mAb of theinvention (i.e., an anti-FGFR2 MAb) is administered to patients having adisease (therapeutic treatment) or at risk of occurrence or recurrenceof a disease (prophylactic treatment). The term “patient” includes humanpatients; veterinary patients, such as cats, dogs and horses; farmanimals, such as cattle, sheep, and pigs; and laboratory animals usedfor testing purposes, such as mice and rats. The methods areparticularly amenable to treatment of human patients. The mAb used inmethods of treating human patients binds to the human FGFR2 protein, thesequence of which is provided by GenBank Locus AF487553. Citations forother FGFRs or FGFs referenced in this disclosure are provided in theBackground section. A mAb to a human protein can also be used in otherspecies in which the species homolog has antigenic crossreactivity withthe human protein. In species lacking such crossreactivity, an antibodyis used with appropriate specificity for the species homolog present inthat species. However, in xenograft experiments in laboratory animals, amAb with specificity for the human protein expressed by the xenograft isgenerally used.

In a preferred embodiment, the present invention provides apharmaceutical formulation comprising the antibodies described herein.Pharmaceutical formulations contain the mAb in a physiologicallyacceptable carrier, optionally with excipients or stabilizers, in theform of lyophilized or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,or acetate at a pH typically of 5.0 to 8.0, most often 6.0 to 7.0; saltssuch as sodium chloride, potassium chloride, etc. to make isotonic;antioxidants, preservatives, low molecular weight polypeptides,proteins, hydrophilic polymers such as polysorbate 80, amino acids suchas glycine, carbohydrates, chelating agents, sugars, and other standardingredients known to those skilled in the art (Remington'sPharmaceutical Science 16^(th) edition, Osol, A. Ed. 1980). The mAb istypically present at a concentration of 0.1-100 mg/ml, e.g., 1-10 mg/mlor 10-50 mg/ml, for example 5, 10, 20, 30, 40, 50 or 60 mg/ml.

In another preferred embodiment, the invention provides a method oftreating a patient with a disease using an anti-FGFR2 mAb in apharmaceutical formulation. The mAb prepared in a pharmaceuticalformulation can be administered to a patient by any suitable route,especially parentally by intravenous infusion or bolus injection,intramuscularly or subcutaneously. Intravenous infusion can be givenover as little as 15 minutes, but more often for 30 minutes, or over 1,2 or even 3 hours. The mAb can also be injected directly into the siteof disease (e.g., a tumor), or encapsulated into carrying agents such asliposomes. The dose given is sufficient to at least partially alleviatethe condition being treated (“therapeutically effective dose”) andoptionally 0.1 to 5 mg/kg body weight, for example 1, 2, 3 or 4 mg/kg,but may be as high as 0.1 or 1 to 10 mg/kg or even 1 to any of 15, 20 or30 mg/kg. A fixed unit dose may also be given, for example, 100, 200,500, 1000 or 2000 mg, or the dose may be based on the patient's surfacearea, e.g., 1000 mg/m². Usually between 1 and 8 doses, (e.g., 1, 2, 3,4, 5, 6, 7 or 8) are administered to treat cancer, but 10, 20 or moredoses may be given. The mAb can be administered daily, biweekly, weekly,every other week, monthly or at some other interval, depending, e.g. onthe half-life of the mAb, for 1 week, 2 weeks, 4 weeks, 8 weeks, 3-6months or longer. Repeated courses of treatment are also possible, as ischronic administration.

A combination of a dose, frequency of administration and route ofadministration effective to at least partially alleviate a diseasepresent in a patient being treated is referred to as therapeuticallyeffective regime. A combination of a dose, frequency of administrationand route of administration effective to inhibit or delay onset of adisease in a patient is referred to as a prophylactically effectiveregime.

Diseases susceptible to treatment with the anti-FGFR2 mAbs of thisinvention include solid tumors, especially those believed to requireangiogenesis or to be associated with detectable or preferably elevatedlevels of FGFR2 and/or an FGF, for example ovarian cancer, endometrialcancer, breast cancer, lung cancer (small cell or non-small cell), coloncancer, prostate cancer, cervical cancer, pancreatic cancer, gastriccancer, esophageal cancer, hepatocellular carcinoma (liver cancer),renal cell carcinoma (kidney cancer), head-and-neck tumors,mesothelioma, melanoma, sarcomas, and brain tumors (e.g., gliomas, suchas glioblastomas). Elevated levels can be measured at the protein ormRNA level in cancerous tissue relative to comparable levels of FGFR2(e.g., FGFR2IIIb) or FGF (e.g., FGF2, FGF7 or FGF10) in normal tissuesuch as tissue-matched noncancerous tissue, preferably from the samepatient. Detectable levels can be similarly measured at the protein ormRNA level in cancerous tissue and compared with background levels incontrol samples in which the analyte (e.g., FGFR2 or FGF) is known to beabsent or relative to negative controls in which detection is performedusing an antibody or primer or probe known not to bind the analyte ornucleic acid encoding the analyte. Leukemias, lymphomas, multiplemyeloma and other hematologic malignancies, especially any of thesecancers having detectable or elevated expression of FGFR2 and/or FGF,can also be susceptible to treatment with the anti-FGFR2 mAbs. Otherdiseases associated with angiogenesis for which treatment with theanti-FGFR2 mAbs of the invention are suitable include age-relatedmacular degeneration (AMD), diabetic retinopathy, neovascular glaucomaand other diseases of the eye; psoriasis and other diseases of the skin;rheumatoid arthritis; and genetic skeletal disorders associated withmutations in the FGFR2, e.g., Apert syndrome, as described above.

In a preferred embodiment, the anti-FGFR2 mAb is administered incombination with (i.e., together with, that is, before, during or after)other therapy. For example, to treat cancer, the anti-FGFR2 mAb may beadministered together with any one or more of the known chemotherapeuticdrugs, for example alkylating agents such as carmustine, chlorambucil,cisplatin, carboplatin, oxaliplatin, procarbazine, and cyclophosphamide;antimetabolites such as fluorouracil, floxuridine, fludarabine,gemcitabine, methotrexate and hydroxyurea; natural products includingplant alkaloids and antibiotics such as bleomycin, doxorubicin,daunorubicin, idarubicin, etoposide, mitomycin, mitoxantrone,vinblastine, vincristine, and Taxol (paclitaxel) or related compoundssuch as Taxotere®; the topoisomerase 1 inhibitor irinotecan; agentsspecifically approved for brain tumors including temozolomide andGliadel® wafer containing carmustine; and inhibitors of tyrosine kinasessuch as Gleevec®, Sutent® (sunitinib malate), Nexavar® (sorafenib) andTarceva® (erlotinib) or Iressa® (gefitinib); inhibitors of angiogenesis;and all approved and experimental anti-cancer agents listed in WO2005/017107 A2 (which is herein incorporated by reference). Theanti-FGFR2 mAb may be used in combination with 1, 2, 3 or more of theseother agents used in a standard chemotherapeutic regimen. Normally, theother agents are those already known to be effective for the particulartype of cancer being treated. The anti-FGFR2 mAb is especially useful inovercoming resistance to chemotherapeutic drugs and thereby increasingtheir effectiveness.

Other agents with which the anti-FGFR2 mAb can be administered to treatcancer include biologics such as monoclonal antibodies, includingHerceptin™ against the HER2 antigen; Avastin® against VEGF; orantibodies to the Epidermal Growth Factor (EGF) receptor such asErbitux® (cetuximab) and Vectibix® (panitumumab). Antibodies againstHepatocyte Growth Factor (HGF) are especially preferred for use with theanti-FGFR2 mAb, including mAb L2G7 (Kim et al., Clin Cancer Res 12:1292,2006 and U.S. Pat. No. 7,220,410) and particularly its chimeric andhumanized forms such as HuL2G7 (WO 07115049 A2); the human anti-HGF mAbsdescribed in WO 2005/017107 A2, particularly 2.12.1; and the HGF bindingproteins described in WO 07143090 A2 or WO 07143098 A2; and otherneutralizing anti-HGF mAbs that compete for binding with any of theaforementioned mAbs. A mAb that binds the cMet receptor of HGF is alsopreferred, for example the anti-cMet mAb OA-5D5 (Martens et al., Clin.Cancer Res. 12:6144, 2006) that has been genetically engineered to haveonly one “arm”, i.e. binding domain. Antibodies against the other FGFRreceptors FGFR1, 3, 4 or against various FGFs such as FGF1, FGF2 andFGF7 are also preferred for use in combination with the anti-FGFR2 mAb.Moreover, the anti-FGFR2 mAb can be used together with any form ofsurgery and/or radiation therapy including external beam radiation,intensity modulated radiation therapy (IMRT) and any form ofradiosurgery such as, e.g. Gamma Knife.

Treatment (e.g., standard chemotherapy) including the anti-FGFR2 mAbantibody may alleviate a disease by increasing the medianprogression-free survival or overall survival time of patients withcancer by at least 30% or 40% but preferably 50%, 60% to 70% or even100% or longer, compared to the same treatment (e.g., chemotherapy) butwithout the anti-FGFR2 mAb, or increase either of these times by 2weeks, 1, 2 or 3 months, or preferably by 4 or 6 months or even 9 monthsor a year. In addition or alternatively, treatment (e.g., standardchemotherapy) including the anti-FGFR2 mAb may increase the completeresponse rate, partial response rate, or objective response rate(complete+partial) of patients with these tumors (e.g., ovarian,gastric, endometrial, pancreatic, breast, lung, colon and glioblastomasespecially when relapsed or refractory) by at least 30% or 40% butpreferably 50%, 60% to 70% or even 100% compared to the same treatment(e.g., chemotherapy) but without the anti-FGFR2 mAb.

Typically, in a clinical trial (e.g., a phase II, phase II/111 or phaseIII trial), the aforementioned increases in median progression-freesurvival and/or response rate of the patients treated with chemotherapyplus the anti-FGFR2 mAb, relative to the control group of patientsreceiving chemotherapy alone (or plus placebo), are statisticallysignificant, for example at the p=0.05 or 0.01 or even 0.001 level. Thecomplete and partial response rates are determined by objective criteriacommonly used in clinical trials for cancer, e.g., as listed or acceptedby the National Cancer Institute and/or Food and Drug Administration.

4. Other Methods

The anti-FGFR2 mAbs of the invention also find use in diagnostic,prognostic and laboratory methods. They may be used to measure the levelof FGFR2 in a tumor or in the circulation of a patient with a tumor, andtherefore to follow and guide treatment of the tumor. For example, atumor associated with high levels of FGFR2 (e.g., increased relative totissue-matched noncancerous sample from the same patient) are especiallysusceptible to treatment with an anti-FGFR2 mAb. In particularembodiments, the mAbs can be used in an ELISA or radioimmunoassay tomeasure the level of FGFR2, e.g. in serum, or in immunohistochemistry tolocalize FGFR2 expression, e.g., in a tumor biopsy specimen. The use oftwo anti-FGFR2 mAbs binding to different epitopes (i.e., not competingfor binding) is especially useful in developing a sensitive “sandwich”ELISA to detect FGFR2. For various assays, the mAb may be labeled withfluorescent molecules, spin-labeled molecules, enzymes or radioisotypes,and may be provided in the form of kit with all the necessary reagentsto perform the assay for FGFR2. In other uses, the anti-FGFR2 mAbs areused to purify FGFR2, e.g., by affinity chromatography.

EXAMPLES Example 1 Reagents and Assays

Preparation of Flag-FGF1, FLAG-FGF2, and FLAG-FGF7. The DNA sequence forhuman FGF1 (the form with 155 amino acids; Chiu et al., Oncogene5:755-1990) and human FGF2 (the form with 155 amino acids; Sommer etal., Biochem. Biophys. Res. Comm. 144:543, 1987) were synthesized(GenScript, Inc), then PCR amplified to have a N-terminal Flag peptidetag and cloned into a derivative of the pET vector (Invitrogen) usingstandard molecular biology techniques. These plasmids were transformedinto E. coli BL21(DE3) cells and FGF1 or FGF2 expression was inducedusing 1 mM IPTG. The level of FGF expression was determined using anFGF1 or FGF2 specific ELISA kit (R&D Systems). FGF was purified usingheparin-Sepharose CL-6B beads (Amersham Biosciences) as described(Wiedlocha et al., Mol. Cell. Biol., 16:270, 1996). Similarly, a genefor human FGF7 (the precursor form with 194 amino acids; Finch, P. W. etal., Science 245:752, 1989) was synthesized and PCR amplified to have aN-terminal Flag tag in a pCMV vector (a derivative of pDrive,Invitrogen), and Flag-FGF10 was made in an analogous way. Plasmid DNAswere transfected into human 293F cells. Culture supernatant of thetransfected 293F cells was used for the ligand-receptor binding assay.

Preparation of FGFR2 fusion proteins. The extracellular domain (ECD) ofhuman FGFR2IIIb and human FGFR2IIIc were expressed as immunoadhesinmolecules. For the alpha forms, the DNA fragments encoding the entireECD of FGFR2IIIb (amino acids 1-378) or FGFR2IIIc (amino acids 1-377)were fused to human Fc (residues 216 to 446) via a polypeptide linker;for the beta forms (missing D1) amino acids 152-378 for FGFR2(beta)IIIband amino acids 152-377 for FGFR2(beta)IIIc were used instead. TheseFGFR2-Fc molecules were expressed by transfecting 293F cells andselecting stable transfectants in the presence of G418 (1 mg/ml) in 293expression medium (Invitrogen). The FGFR2-Fc secreted from 293Ftransfected cells was purified using a protein A/G column. Similarly,cDNA of the cynomolgus (cyno) monkey FGFR2 ECD was cloned by standardtechniques from cyno liver mRNA, and amino acids 1-378 were fused tohuman Fc to create cyno FGFR2IIIb-Fc for expression. ChimpanzeeFGFR2IIIb-Fc was constructed by using in vitro mutagenesis to convertthe one amino acid in the human FGFR2IIIb ECD that differs from chimpFGFR2IIIb into the chimp amino acid (residue 186 methionine tothreonine, based on the known sequences in GenBank). MouseFGFR2(beta)IIIb-Fc protein was purchased from R&D Systems (Catalog #708-MF).

ELISA assay for mAb binding to FGFR2 fusion protein. ELISA plates werecoated with goat anti-human IgG-Fc (2 μg/ml) overnight at 4° C. Thennonspecific binding sites were blocked with 2% BSA for 1 hr at RT.Plates were incubated with one of the FGFR2 fusion proteins describedabove (1 μg/ml) for 1 hr, followed by incubation with variousconcentrations of mAbs or hybridoma culture fluids for 1 hr. The boundmAb was detected with HRP-Goat anti-mouse antibody followed by washing,addition of TMB substrate (Sigma) and reading at 450 nm. In all ELISAassays, plates were washed 3 times between each step.

Flow cytometry. After washing twice in cell sorting buffer (CSB:PBS/1%FBS/0.02% NaN₃), 2×10⁵ cells were resuspended in 50 μl of CSB in amicrotiter well and incubated with 50 μl of the anti-FGFR2 mAb to betested (1 μg/50 μl) for 1 hr on ice. Cells were then washed twice in CSBand the bound antibodies detected by incubation with FITC-goatanti-mouse IgG (Jackson ImmunoResearch Laboratories) for 1 hr on ice.After washing twice in CSB, cells were analyzed on a FACScan (BectonDickinson).

Example 2 Generation of Monoclonal Antibodies to FGFR2

Balb/c mice (5-6 week old female) were immunized by injection in theirrear footpads at 1 week intervals either 20 or 22 times withFGFR2(beta)IIIb-Fc (initial dose 10 μg/footpad, then 5 μg/footpad), orwith 17 doses FGFR2IIIc-Fc (initial dose 10 μg/footpad, then 5 doses at2 μg, then at 5 μg) followed by 5 doses FGFR2(beta)IIIc-Fc (at 5μg/footpad), with the antigen suspended in MPL/TDM (Sigma-Aldrich).Three days after the final injection, popliteal lymphoid cells wereextracted and fused with P3/X63-Ag8U1 mouse myeloma cells at a 1:1 ratiousing a Hybrimune Electrofusion System (Cyto Pulse Sciences). Hybridomaswere selected by the addition of 2x HAT (Sigma) 24 hr later. Ten daysafter the fusion, hybridoma culture supernatants were screened for theirability to bind to FGFR2IIIb-Fc but not to human IgG using ELISA.Selected mAbs were then screened for their ability to recognizeFGFR2IIIb on the human gastric tumor cell line SNU-16 (Shin et al, J.Cancer Res. Clin. Oncol. 126:519, 2000). Selected hybridomas were thencloned twice using the limiting dilution technique. Three mAbs selectedin this way were GAL-FR21 and GAL-FR22 from the first immunizationregime, and GAL-FR23 from the second immunization regime. Properties ofthese mAbs are shown in FIG. 2 as further described below.

In addition, a number of other anti-FGFR2 mAbs were obtained from thefusions, including FR2bB 100.12.9, FR2bC 54.8.11, FR2bC 100.7.9, FR2bC101.8.2, FR2bC 115.1.5, FR2bC 149.8.8, FR2bB 11.5.3, and FR2bB 18.1.6.

Example 3 Properties of the Anti-FGFR2 mAbs

As seen in FIG. 3, all three selected mAbs GAL-FR21, GAL-FR22 andGAL-FR23 bind well to FGFR2IIIb in the ELISA assay described inExample 1. By using the four different forms of FGFR2-Fc in the ELISA,it was determined that each of these mAbs has a different bindingpattern and therefore epitope (FIG. 4). GAL-FR21 binds to both the alphaand beta forms of FGFR2IIIb (i.e., with and without D1), but not toFGFRIIIc. The epitope therefore cannot involve D1 and must involveD3111b, so is likely encompassed within D311Ib or D3. GAL-FR22 alsobinds to both alpha and beta forms, but in both the IIIb and IIIccontext, so the epitope is presumably encompassed in D2-D311Ia orcertainly D2-D3. Finally, GAL-FR23 does not bind to either of the betaforms, so its epitope must be wholly or partially in D1. Hence, mAbsthat have an epitope either in D1 or D2-D3 or D3 are encompassed in theinstant invention.

To confirm that the mAbs GAL-FR21, GAL-FR22 and GAL-FR23 bind todifferent epitopes, a competition experiment was performed in which eachmAb was biotinylated, and then 0.4 μg of the biotinylated mAb wascompeted with a 100:1 excess of each of the other unlabeled mAbs (orcontrol murine mAb 5G8) for binding to FGFR2IIIb-Fc in the ELISA assaydescribed above (but with HRP-streptavidin as the detection reagent). Asseen in FIG. 5, each mAb competed with itself for binding but not withthe other mAbs, showing that they have different epitopes. In addition,the other mAbs FR2bB 100.12.9, FR2bC 54.8.11, FR2bC 100.7.9, FR2bC101.8.2, Fr2bC 115.1.5, FR2bC 149.8.8 competed for binding withbiotinylated GAL-FR21 in this assay so have the same or overlappingepitope as GAL-FR21, while the mAbs FR2bB 11.5.3, and FR2bB 18.1.6competed for binding with biotinylated GAL-FR22 so have the same oroverlapping epitope as GAL-FR22.

To confirm that the selected mAbs bind to the appropriate forms of FGFR2on the cell membrane, flow cytometry was employed. KATO-III (ATCCHTB-103) and SNU-16 (ATCC CRL-5974) cells, which overexpress FGFR2IIIb,were used to test binding to that form of the receptor. As seen in FIG.6, all three mAbs GAL-FR21, GAL-FR22 and GAL-FR23 bind both cell lines,as expected from their epitopes described above. Human 293F cellstransfected with a gene for FGFRIIIc were used to test binding to thatform, after verifying that none of the mAbs bind to the host 293F cellsthemselves. As seen in FIG. 7, GAL-FR22 and GALFR23 but not GALFR21 bindto the FGFRIIIb-transfected cells, as expected from their epitopes.Finally, since the S252W mutation of FGFR2 is found in some cancercells, binding of the mAbs to 293F cells transfected with an FGFR2IIIbgene constructed to contain that mutation (FGFR2IIIb(S252W)) was tested.As also seen in FIG. 7, all the mAbs bound to theFGFR2IIIb(S252W)-transfected cells. The ability to bind FGFR2IIIb(S252W)is a preferred property of mAbs of the invention.

To determine the ability of the mAbs to inhibit binding of FGF ligandsto FGFR2, an ELISA assay was used. ELISA wells were coated with 2 μg/mlof goat anti-human IgG-Fc overnight at 4° C. After blocking with 2% BSAfor 1 hr at RT, the wells were incubated with 0.5 μg/ml of FGFR2IIIb-Fcfor 1 hr, followed by incubation with Flag-FGF1 or Flag-FGF2 (0.2 μg/ml)in the presence of various concentrations of mAbs for 1 hr. The boundFlag-FGF was detected by the addition of HRP-anti-Flag M2 antibody(Sigma) and then addition of TMB substrate. As can be seen from FIG. 8A,the mAb GAL-FR21 weakly blocked binding of FGF1 to FGFR2IIIb in thisassay, but GAL-FR22 and GAL-FR23 did not block FGF1 binding. In contrastGAL-FR21 strongly blocked binding of FGF2 to FGFR2IIIb, GAL-FR22moderately blocked FGF2 binding, and GAL-FR23 did not block binding. Insimilar assays but using Flag-FGF7 and Flag-FGF10, it was also shown(FIG. 8B) that GAL-FR21 and GAL-FR22 block binding of FGF7 and FGF10 toFGFR2IIIb. Indeed, advantageous mAbs of the invention, like GAL-FR21 andGAL-FR22, block binding of FGF2 and FGF7 and/or FGF10 to FGFR2IIIb,preferably by 80% or 90% or 95% or completely or essentially completely.Hence GAL-FR21 and GAL-FR22 but not GAL-FR23 have been shown toneutralize at least one biological activity of FGFR2.

Example 4 Xenograft Models

Xenograft experiments are carried out as described previously (Kim etal., Nature 362:841,1993). Human tumor cells typically grown in completeDMEM medium are harvested in HBSS. Female athymic nude mice or NIH-IIIXid/Beige/nud mice (4-6 wks old) are injected subcutaneously with2-10×10⁶ cells in 0.1 ml of HBSS in the dorsal areas. When the tumorsize reaches 50-100 mm³, the mice are grouped randomly and 5 mg/kg (100μg total) or some other dosage of mAbs are administered i.p. twice perweek in a volume of 0.1 ml. Tumor sizes are determined twice a week bymeasuring in two dimensions [length (a) and width (b)]. Tumor volume iscalculated according to V=ab²/2 and expressed as mean tumor volume±SEM.The number of mice in each treatment group is typically 5-7 mice.Statistical analysis can be performed, e.g., using Student's t test.

FIGS. 9 and 10A show that in various experiments GAL-FR21, GAL-FR22 andGAL-FR23 administered at a dose level of 20 μg (1 mg/kg) twice per weekall strongly inhibited the growth of SNU-16 gastric tumor xenografts,with GAL-FR21 being most potent and completely inhibiting growth of thexenograft. The mAb FR2bC 54.8.11 mentioned above that competes forbinding with GAL-FR21 also inhibited xenograft growth. FIG. 10B showsthat GAL-FR21 and GAL-FR22 administered at a dose level of 50 μg (2.5mg/kg) twice per week also strongly inhibited growth of xenografts ofthe OCUM-2M human gastric tumor cell line (which is described in Yashiroet al., Jpn J Cancer Res 85:883, 1994). The ability of the mAbs toinhibit xenografts of KATO III or other FGFR2-expressing cell lines isshown similarly. The ability of the mAbs to inhibit tumor growthadditively or synergistically with other anti-tumor agents as describedabove is demonstrated by treating groups of xenografted mice with themAb alone, the other agent alone, and the mAb together with the otheragent, and noting that treatment with both agents has a greaterinhibitory effect than either agent alone.

Example 5 Binding of mAbs to FGFR2 from Other Species

To determine the ability of the mAbs to bind to FGFR2 from species otherthan human, ELISA assays were used. ELISA wells were coated with 2 μg/mlof goat anti-human IgG-Fc overnight at 4° C. After blocking with 2% BSAfor 1 hr at RT, the wells were incubated with 0.2 μg/ml of FGFR2IIIb-Fcfor 1 hr, where the FGFRIIIb in the fusion protein was either human,mouse, cynomolgus monkey or chimpanzee FGFRIIIb. The wells were thenincubated with various concentrations of GAL-FR21 or GAL-FR22 mAb. Thebound mAbs were detected by addition of HRP-conjugated goat anti-mouseIgG-Fc and then TMB substrate. FIG. 11 shows that GAL-FR21 binds tomouse FGFR2 almost as well (within 10-fold) as human FGFR2, whileGAL-FR22 binds to mouse FGFR2 moderately well (within about 100-fold ofhuman FGFR2). FIG. 12 shows that GAL-FR21 binds to cynomolgus monkeyFGFR2 as well as (indistinguishably from) human FGFR2, while GAL-FR22binds to cynomolgus monkey FGFR2 moderately well (within about 100-foldof human FGFR2). A similar experiment with chimpanzee FGFR2 gave thesame results as with cynomolgus monkey FGFR2: GAL-FR21 bound tochimpanzee FGFR2 as well as (indistinguishably from) human FGFR2, whileGAL-FR22 bound to chimpanzee FGFR2 moderately well (within about100-fold of human FGFR2). Preferred mAbs of the invention, like GAL-FR21and GAL-FR22, bind to all of mouse, monkey, chimpanzee and human FGFR2,and most preferably bind mouse FGFR2 within 2, 10, 100 or 1000 fold aswell as human FGFR2, and/or bind monkey and/or chimpanzee FGFR2 within2, 10 or 100-fold or indistinguishably from (within experimentalvariation) human FGFR2 (as measured, e.g., by K_(a)). Binding to FGFR2from such other species makes testing of the mAbs in those animalspecies easier to conduct.

Example 6 Humanization of GAL-FR21 and GAL-FR22

Cloning of the light and heavy chain variable regions of the GAL-FR21mAb, construction and expression of a chimeric mAb, and design,construction, expression and purification of a humanized GAL-FR21 mAbwere all performed using standard methods of molecular biology, e.g. asdescribed in US 20080019974 for the L2G7 mAb, which is hereinincorporated by reference for all purposes. The amino acid sequences ofthe (mature) light and heavy chain variable (V) regions of GAL-FR21 areshown respectively in FIGS. 13A and 13B, top lines labeled GAL-FR21.More specifically, to design a humanized GAL-FR21 mAb, the methods ofQueen et al., U.S. Pat. Nos. 5,530,101 and 5,585,089 were generallyfollowed. The human V_(K) sequence CAG27369 and VH sequence AAB00780, asshown respectively in FIGS. 13A and 13B, bottom lines, were respectivelychosen to serve as acceptor sequences for the GAL-FR21 VL and VHsequences because they have particularly high framework homology (i.e.,sequence identity) to them. A computer-generated molecular model of theGAL-FR21 variable domain was used to locate the amino acids in theGAL-FR21 framework that are close enough to the CDRs to potentiallyinteract with them. To design the humanized GAL-FR21 light and heavychain variable regions, the CDRs from the mouse GAL-FR21 mAb were firstconceptually grafted into the acceptor framework regions. At frameworkpositions where the computer model suggested significant contact withthe CDRs, which may be needed to maintain the CDR conformation, theamino acids from the mouse antibody were substituted for the humanframework amino acids. For the humanized GAL-FR21 mAb designatedHuGAL-FR21, this was done at residues 27, 28, 30 (within Chothiahypervariable loop H1) and 48 and 67 of the heavy chain and at noresidues in the light chain, using Kabat numbering. The light and heavychain V region sequences of HuGAL-FR21 are shown in FIGS. 13A and 13Brespectively, middle lines labeled HuGAL-FR21, where they are alignedagainst the respective GAL-FR21 donor and human acceptor V regions—theCDRs (as defined by Kabat) are underlined and the substituted aminoacids listed above are double-underlined.

The invention provides not only a humanized GAL-FR21 mAb, HuGAL-FR21,including the light and heavy chain V regions shown in FIG. 13, but alsovariant humanized mAbs whose light and heavy chain variable regionsdiffer from the sequences of HuGAL-FR21 by a small number (e.g.,typically no more than 1, 2, 3, 5 or 10) of replacements, deletions orinsertions, usually in the framework but possibly in the CDRs. Inparticular, only a subset of the substitutions described above can bemade in the acceptor frameworks, or additional substitution(s) can bemade, e.g., the mouse GAL-FR21 VH amino acid 69 L may replace theacceptor amino acid 691, and/or the mouse amino acids may replace therespective amino acids in the humanized light chain at any of theKabat-numbered positions 1, 3 and 60 and 63, which have some proximityto the CDRs. Indeed, many of the framework residues not in contact withthe CDRs in the humanized mAb can accommodate substitutions of aminoacids from the corresponding positions of the donor mouse mAb or othermouse or human antibodies, and even many potential CDR-contact residuesare also amenable to substitution or even amino acids within the CDRsmay be altered. One example of a CDR substitution is to substitute aresidue in a CDR with the residue occupying the corresponding positionof the human acceptor sequence used to supply variable regionframeworks.

Most often the replacements made in the variant humanized GAL-FR21sequences are conservative with respect to the replaced HuGAL-FR21 aminoacids. Amino acids can be grouped as follows for determiningconservative substitutions, i.e., substitutions within a group: Group I(hydrophobic sidechains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe.

Preferably, replacements in HuGAL-FR21 (whether or not conservative)have no substantial effect on the binding affinity or potency of thehumanized mAb, that is, its ability to neutralize the biologicalactivities of FGFR2 (e.g., the potency in some or all of the assaysdescribed herein of the variant humanized GAL-FR21 mAb is essentiallythe same, i.e., within experimental error, as that of HuGAL-FR21).Preferably the mature variant light and heavy chain V region sequencesare at least 90%, more preferably at least 95%, and most preferably atleast 98% identical to the respective HuGAL-FR21 mature light and heavychain V regions. Alternatively, other human antibody variable regionswith high sequence identity to those of GAL-FR21 are also suitable toprovide the humanized antibody framework, especially kappa V regionsfrom human subgroup I and heavy chain V regions from human subgroup I,or consensus sequences of these subgroups.

In other humanized antibodies, at least 1, 2, 3, 4, or all 5 of thepositions of acceptor to donor substitutions mentioned in connectionwith the exemplified antibody (i.e., H27, H28, H30, H48, H67) arepreferably occupied by the residue occupying the corresponding positionof the mouse donor antibody heavy chain. If the heavy chain acceptorsequence is other than AAB00780, an acceptor to donor substitution mayor may not be required for the specified occupancy of a particularvariable framework region position depending on whether the residueoccupying the specified position is already the same between theacceptor and donor.

The exemplary mAb HuGAL-FR21 discussed here has human K and y1 constantregions, e.g., as presented in US 20080019974, and is therefore an IgG1.The complete sequences of the (mature) light and heavy chains ofHuGAL-FR21 are shown in FIG. 14. While these sequences are respectivelyof the Km(3) and G1m(3) allotypes, it is understood that IgG1 mAbs ofany (IgG1, K) allotype are encompassed by the designation HuGAL-FR21. Itwill also be understood that when HuGAL-FR21 is manufactured byconventional procedures, one to several amino acids at the amino orcarboxy terminus of the light and/or heavy chain, such as the C-terminallysine of the heavy chain, may be missing or derivatized in a proportionor all of the molecules, and such a composition will still beencompassed by the designation HuGAL-FR21 and considered a humanizedGAL-FR21 mAb. Humanized mAbs of other isotypes (e.g., IgG2, IgG3 andIgG4) can be made by combining the HuGAL-FR21 variable regions with theappropriate human constant regions. Replacements can be made in theHuGAL-FR21 constant regions to reduce or increase effector function suchas complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al.,U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazaret al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolonghalf-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213,2004). Specifically but without limitation, HuGAL-FR21 having mutationsin the IgG constant region to a Gln at position 250 and/or a Leu atposition 428 are embodiments of the present invention.

To compare the binding affinity of HuGAL-FR21 with that of the mouse mAbGAL-FR21, a competitive binding experiment was performed using standardELISA technology. Specifically, ELISA wells were coated with 2 μg/ml ofgoat anti-human IgG-Fc overnight at 4° C. After blocking with 2% BSA for1 hr at RT, the wells were incubated with 0.5 μg/ml of FGFR2IIIb-Fc. Thewells were then incubated with biotinylated GAL-FR21 mAb (0.05 μg/ml) inthe presence of increasing concentrations of unlabeled GAL-FR21,HuGAL-FR21 or control human antibody hIgG. The level of biotinylatedGAL-FR21 bound was determined by the addition of HRP-streptavidin andsubstrate. As shown in FIG. 15, HuGAL-FR21 and GAL-FR21 competedapproximately equally well, with HuGAL-FR21 possibly slightly better,indicating that the binding affinity for FGFR2 of HuGAL-FR21 is at leastas high as (mouse) GAL-FR21 mAb. From the concentration of HuGAL-FR21required to inhibit binding of the labeled mAb by 50%, one may estimatethat the binding affinity K_(a) of HuGAL-FR21 for FGFR2 is at leastapproximately 10⁹ M⁻¹. HuGAL-FR21 may also be tested in any of thebiological assays for FGFR2 activity described herein, e.g., inhibitionof binding of FGF2 or FGF7 to FGFR2, and will inhibit FGFR2 activitycomparably to GAL-FR21.

A humanized GAL-FR22 mAb can be designed, constructed, produced andassayed in the same or similar way as HuGAL-FR22. The amino acidsequences of the (mature) light and heavy chain variable regions ofGAL-FR22 are shown respectively in FIGS. 16A and 16B, lines labeledGAL-FR22. A humanized GAL-FR22 mAb has a humanized light chaincomprising CDRs from the sequence in FIG. 16A and a humanized heavychain comprising CDRs from the sequence of FIG. 16B. In some instances,the humanized GAL-FR22 mAb comprises the three light chain CDRs shown inFIG. 16A and the three heavy chain CDRs shown in FIG. 16B. Preferably,the humanized GAL-FR22 mAb has binding affinity for FGFR2 within 2 or 3fold of the affinity of the mouse GAL-FR22 mAb, and most preferably hasbinding affinity indistinguishable or greater than the affinity of theGAL-FR22 mAb, as measured, e.g., by a competition ELISA as described forHuGAL-FR21 and GAL-FR21.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the invention. Unlessotherwise apparent from the context any step, element, embodiment,feature or aspect of the invention can be used with any other. Allpublications, patents, patent applications, accession numbers and thelike cited are herein incorporated by reference in their entirety forall purposes to the same extent as if each individual publication,patent, patent application or accession number was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes. If a nucleic acid or protein sequence associated withan accession number is changed, the version of the sequence associatedwith that accession number as of Nov. 7, 2008 is intended.

The hydridomas producing the monoclonal antibodies GAL-FR21, GAL-FR22,and GAL-FR23 have been deposited at the American Type CultureCollection, P.O. Box 1549 Manassas, Va. 20108, as respectively ATCCNumbers PTA-9586 on Nov. 6, 2008, PTA-9587 on Nov. 6, 2008 and PTA-9408on Aug. 12, 2008, under the Budapest Treaty. These deposits will bemaintained at an authorized depository and replaced in the event ofmutation, nonviability or destruction for a period of at least fiveyears after the most recent request for release of a sample was receivedby the depository, for a period of at least thirty years after the dateof the deposit, or during the enforceable life of the related patent,whichever period is longest. All restrictions on the availability to thepublic of these cell lines will be irrevocably removed upon the issuanceof a patent from the application.

1. A humanized or human monoclonal antibody (mAb) that binds fibroblastgrowth factor receptor 2 (FGFR2) and inhibits growth of a human tumorxenograft in a mouse.
 2. The mAb of claim 1 which inhibits binding ofFGF2 to FGFR2.
 3. The mAb of claim 1 which binds to FGFR2IIIb but notFGFR2IIIc.
 4. The mAb of claim 1 which binds to both FGFR2IIIb andFGFR2IIIc.
 5. The mAb of claim 1 which is a Fab or F(ab′)2 fragment orsingle-chain antibody.
 6. A pharmaceutical composition comprising a mAbof claim
 1. 7. A method of treating cancer in a patient comprisingadministering to the patient a pharmaceutical composition comprising themAb of claim
 1. 8. A monoclonal antibody (mAb) that competes for bindingto FGFR2 with an antibody selected from the group of GAL-FR21, GAL-FR22and GAL-FR23, wherein the mAb is genetically engineered.
 9. The mAb ofclaim 8 which inhibits growth of a SNU-16 human tumor xenograft in amouse.
 10. The mAb of claim 8 which is chimeric or humanized.
 11. ThemAb of claim 8 which is a humanized GAL-FR21 or GAL-FR22 mAb.
 12. ThemAb of claim 8 which is human.
 13. A pharmaceutical compositioncomprising a mAb of claim
 8. 14. A method of treating cancer in apatient comprising administering to the patient a pharmaceuticalcomposition comprising the mAb of claim
 8. 15. The method of claim 14wherein said cancer is gastric cancer.
 16. A humanized antibodycomprising a humanized light chain comprising CDRs from the sequence inFIG. 13A (GAL-FR21; SEQ ID NO:1) and a humanized heavy chain comprisingCDRs from the sequence of FIG. 13B (GAL-FR21; SEQ ID NO:4), orcomprising a humanized light chain comprising CDRs from the sequence inFIG. 16A (GAL-FR22; SEQ ID NO:7) and a humanized heavy chain comprisingCDRs from the sequence of FIG. 16B (GAL-FR22; SEQ ID NO:8).
 17. Ahumanized antibody of claim 16 comprising the three light chain CDRsshown in FIG. 13A (GAL-FR21; SEQ ID NO:1) and the three heavy chain CDRsshown in FIG. 13B (GAL-FR21; SEQ ID NO:4), or comprising the three lightchain CDRs shown in FIG. 16A (GAL-FR22; SEQ ID NO:7) and the three heavychain CDRs shown in FIG. 16B (GAL-FR22; SEQ ID NO:8).
 18. A humanizedantibody of claim 17 wherein the light chain variable region has atleast 95% sequence identity to the sequence shown in FIG. 13A(HuGAL-FR21; SEQ ID NO:9) and the heavy chain variable region has atleast 95% sequence identity to the sequence shown in FIG. 13B(HuGAL-FR21; SEQ ID NO:10).
 19. The humanized antibody of claim 18wherein residues H27, H28, H30, H48, and H67 by Kabat numbering areoccupied by the residue occupying the corresponding position of theheavy chain shown in FIG. 13B (GAL-FR21; SEQ ID NO:4).
 20. The humanizedantibody of claim 19 wherein the light chain variable region has thesequence shown in FIG. 13A (HuGAL-FR21; SEQ ID NO:9) and the heavy chainvariable region has the sequence shown in FIG. 13B (HuGAL-FR21; SEQ IDNO:10).